CRISPR/Cas9 mediated genome editing has been widely used as a powerful tool for biological and therapeutic applications. Adeno-associated virus (AAV) derived vectors and DNA nanoclews were reported to deliver plasmid-encoding CRISPR/Cas in a wide variety of cells and animal models. Yet, potential off-target effects are significant safety issues if Cas9 is present for long term. One alternative approach is to express Cas9 protein using Cas9 mRNA; however, efficient delivery of Cas9 mRNA is formidable due to its large size (up to 4.5 k nucleotides), high density of negative charges, and weak tolerance of enzymes in serum. Hence, new biomaterials are needed to overcome this obstacle and enable broad applications for delivering nucleic acids, for example, nucleic acids for use in the CRISPR/Cas9 gene editing system.
Messenger RNA (mRNA) based therapeutics have shown great promise for expressing functional antibodies and proteins. Clinical studies have explored mRNA for use as vaccines through local administration of naked mRNA or mRNA-transfected dendritic cells in order to induce antigen-specific immune responses. Recently, extensive efforts have been devoted to achieving the systemic delivery of mRNA using liposomes, polymeric nanoparticles, and mRNA-protein complexes. Although significant advances have been made, new mRNA carriers are needed in order to improve delivery efficiency and maximize therapeutic windows of mRNA therapeutics in different human conditions.
Lipid and lipid-like nanoparticles (LNPs and LLNs) represent one type of biomaterial for efficient delivery of RNAs including siRNA, miRNA, and mRNA. Recently, a few lipid-like nanoparticle-based delivery systems demonstrated efficient delivery of mRNAs for expression of functional proteins including factor IX, erythropoietin and Cas9. However, these previous lipid-like compounds are not considered to be biodegradable, which may lead to potential side effects. Therefore, what is needed are materials with biodegradable bonds that enable rapid elimination from plasma and tissues, and that substantially improve their tolerability in preclinical studies.
The compounds, compositions, and methods disclosed herein address these and other needs.